Linhagens de células de melanoma: Mutações e impacto em vias de transdução de sinal / Melanoma cell lines: Mutations and impact on signal transduction pathways

Linhagens de células tumorais são amplamente utilizadas em pesquisa, uma vez que são úteis para estudos iniciais visando diferentes propósitos, tais como triagem de compostos, ensaios de migração, proliferação, morte celular, expressão gênica, identificação de proteínas específicas, vias de sinalização, dentre outras. Neste aspecto, o conhecimento de características moleculares das linhagens pode auxiliar no planejamento experimental e na interpretação da resposta frente ao estímulo de morte em virtude de um tratamento. Nesta revisão, enfocamos nas alterações moleculares e seus impactos em vias de sinalização, em linhagens de melanoma mais comumente utilizadas como modelos para estudos in vitro. Portanto, essas informações podem ser úteis para ajudar na decisão do modelo celular mais adequado para determinada pergunta científica

Plasticidade molecular da matriz extracelular contribui para metástase e resistência a terapias / Molecular plasticity of the extracellular matrix contributes to metastasis and resistance to therapies

A matriz extracelular (MEC) trata-se de um componente não celular presente em todos os tecidos e órgãos e atua não apenas como uma estrutura física importante para os constituintes celulares, mas também como ponto de partida para eventos bioquímicos e biomecânicos essenciais para a morfogênese, diferenciação e homeostase do tecido. A MEC é composta de um conjunto complexo de proteínas fibrosas, proteoglicanos e outras moléculas, tais como citocinas, fatores de crescimento e hormônios, cuja composição varia de tecido para tecido e é alterada frente a diferentes condições fisiológicas (renovação e reparo tecidual) e associadas às doenças, incluindo câncer, razão pela qual componentes da MEC são denominados como Hallmarks do câncer. Neste contexto, a remodelação da MEC é uma das estratégias que os tumores utilizam para criar um microambiente que promove a tumorigênese e metástase através de diferentes mecanismos. Nesta revisão as características funcionais da MEC serão abordadas e também destacado o entendimento atual dos mecanismos físicos, celulares e moleculares pelos quais a MEC do tumor afeta a eficiência da quimioterapia, radioterapia e imunoterapia. 

Riboflavin: a multifunctional vitamin

Riboflavin, a component of the B2 vitaminic complex, plays important roles in biochemistry, especially in redox reactions, due to the ability to participate in both one- and two-electron transfers as well as acting as a photosensitizer. Accordingly, low intakes of this vitamin have been associated with different diseases, including cancer and cardiovascular diseases. Riboflavin is thought to contribute to oxidative stress through its capacity to produce superoxide but, interestingly, it can also promote the reduction of hydroperoxides. This peculiar and multifunctional behavior allows riboflavin to take part in various biochemical pathways as a nucleophile and an electrophile, turning it into a versatile and important biological compound.887891Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Herregårde – fornyere på landet? Gram Slot A/S mellem oplevelsesøkonomi og kulturarv

Denne artikel tager udgangspunkt i en opfattelse af, at historiske bygninger og kulturlandskaber kan medvirke til at skabe dynamik og fornyelse i lokalsamfund på landet præget af stilstand og tilbagegang. Eksempelvis var Fonden Realdania’s kampagne “Fremtidens Herregård” begrundet i et udtalt ønske om, at herregårdene kunne bidrage til regional udvikling på landet. Undersøgelser af kommercielle aktiviteter og oplevelsesøkonomi på Gram Slot A/S og andre danske herregårde vil demonstrere, på hvilke måder herregårdenes fortidige virkelighed finder anvendelse i nutidig virksomhedsdrift. Analyserne fører frem til en principiel diskussion af, i hvilket omfang – og med hvilke formål – kulturarv kan fungere som en relevant ressource for lokal og regional udvikling.SummaryCommercial heritage management at Gram Slot A/SIn recent decades, a substantial number of Danish country houses [da: “herregårde”] have explored business opportunities within the so-called “experience economy”. Privately owned country houses comprise a unique field of investigation, because these estates constitute both a shared cultural heritage and modern business ventures. In their efforts to generate income to uphold the country house and estate, owners and managers welcome visitors onto the estate and into the country house. Paying visitors are invited to “experience the country house” as venues for, among other things, weddings, parties, conferences, concerts, Christmas fairs, historical festivals, team-building, golf and tourism. This article comprises an analysis of commercial offerings and visitor practices at Gram Slot A/S, a Danish country house in southern Jutland operating as a private limited company since 2007. Gram Slot A/S produces organic food such as milk, oatmeal and potatoes as well as tourist services like fairs, dinner parties and holiday visits. Guided tours in the country house are provided to visitors with interpretations of Grams’ history as well as the present business operations. An analysis of six markedly different guided tours at Gram showed how different guides and different audiences could result in surprising shifts of perspective. Gram was variously presented as a historical country house, an innovative family business, an organic farm, a regional centre and a place of unique aesthetic qualities. The guided tours created a strong narrative that presented Gram as a place of past, present and future – a reminder that new activities may also create new history and new meaning(s) to country houses and other heritage sites. Through a presentation of various understandings of “cultural heritage” the article discusses commercial offerings and “experiences” in heritage management. The analysis shows that commercial heritage management may help bring revenue for conservation and provide historical “feeling” and a sense of place to visitors and to local and national communities. However, by academic standards historical representations are often flawed and rarely adds to historical understanding. In the final analysis, the article advocates a pragmatic view on cultural heritage management, in which country houses by adapting to new challenges – as cultural heritage as well as contemporary business – may create new meaning to historical buildings and thereby offer a different and rewarding view on how we relate to our past

Caracterização Cinetica e Fisico-quimica de fosfatase acidas purificadas de sementes de soja

Orientador: Hiroshi AoyamaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As quatro isofonnas de fosfatases ácidas purificadas de sementes de soj:quiescentes foram caracterizadas do ponto de vista fisico-químico e cinético. A isofonnas AP1, AP2, AP3A e AP3B apresentaram pontos isoelétricos de 4,9, 5,7 9,8 e 7,4, e quantidade de carboidratos totais de 30, 25, 18 e 10%, respectivamente Somente as isoformas AP 1 e AP3A apresentaram feITo em suas estruturas. As 4 isoformas apresentaram alta atividade catalítica à 80°C quando o pNPP foi utilizado como substrato, no entanto, quando TyrP foi utilizada, esta temperatura foi de 60 °C e para o PPi a temperatura máxima foi em tomo de 60°C para as isoformas AP 1 AP2 e AP3B e de 50°C para a isoformas AP3A. Estudos de estabilidade térmica foram realizados pré incubando-se as isoformas na ausência do substrato. Todas as isoformas perderam 20% da atividade quando pré incubadas a 60°C por 60 min. A 70°C as isoformas AP3A e AP3B mantiveram 30% de atividade residual. Todas a isoformas foram inativadas quando pré incubadas a 80°C. Estudos de estabilidade térmica na presença de alguns possíveis protetores, mostraram que os produtos da reação com o pNPP (pNP e fosfato) eram responsáveis pela estabilização de toda isoformas, favorecendo a alta atividade catalítica a 80°C. As 4 isoformas não foram significativamente afetadas por compostos que interagem com grupos -SH (pCMB, H2O2, CU²+), indicando a ausência de tais grupos no sítio ativo. Piridoxal fosfato também não apresentou efeito inibitório, (que mostra que estas enzimas não contêm resíduos de lisina essenciais para catálise. Todas as isoformas foram inibidas competitivamente pelo fosfato, vanadatc pervanadato, fluoreto e molibdato. A determinação das constantes de inibição (Ki revelou que o molibdato é o inibidor mais potente para todas as isoformas. A ConA apresentou efeito ativador somente para a isoformas API, o qual era dependente d4 substrato utilizado. As isoformas AP 1 e AP2 mostraram uma maior especificidade por substrato: que AP3A e AP3B. Foram realizados estudos cinéticos detalhados utilizando-S4 PEP, PPi, TyrP, Glic6P e Frut6P como substratos. Os resultados cinético: mostraram que estas isoformas poderiam participar em diferentes rotas metabólicasAbstract: The four isoforms of acid phosphatase (AP1, AP2, AP3A and AP3B) purified from mature soybean seeds, had their physico-chemical and kinetil properties detennined. The APl, AP2, AP3A and AP3B isofonns presentel isoeIectric points of 4.9, 5.7, 9.8 and 7.4 and total carbohydrate of 30, 25, 18 anl 10%, respectiveIy. Among the four isoforms only APl and AP3A presented iron u their structures. The four isoforms of soybean seeds acid phosphatases presentel high activities, even at temperatures above 80°C when pNPP was utilized a: substrate. On the other hand, Iowest optimum temperature values were obtainec using other substrates. With tyrosine phosphate, the maximum activity was observec at 60°C, and when PPi was used as substrate, the optimum temperature values wen 60°C for APl, AP2 and AP3B, and 50°C for AP3A. In order to investigate the thermal stability of the four acid phosphatases, the enzymes were preincubated it the absence of substrate. The four isoforms Iost only 20% activity at 60°C, afier 60 min; the isoforms AP3A and AP3B retained 30% of activity at 70°C, after 60 min; alI the isoforms were inactivated at 80°C, after 5 min. In order to verify whether the enzymatic forms could be protected against thermal inactivation, we tested some compounds with different characteristics. The best protective effect was observec when the enzymes were incubated in the presence of both reaction products, p-nitrophenoI and inorganic phosphate. The soybean seeds acid phosphatases were not significantly affected by compounds that interacted with -SH residues (pCMB, H2O2, CU²+), indicating th absence of such residues in the active sites. Pyridoxal phosphate had no effecl suggesting that lysine residues were not important to the enzyme catalysis. The four acid phosphatase isoforms API, AP2, AP3A and AP3B wer competitively inhibited by phosphate, vanadate, molybdate and fluoride; inhibitio: constant studies showed that molybdate was the most potent inhibitor. The AP soform was activated by lectin (ConA), which effect was dependent on the substrat utilized. The AP 1 and AP2 isoforms showed higher substrate specificity whe: compared with AP3A and AP3B. Detailed kinetic properties were studied using PEP, PPi, TyrP, Gluc6P an, Frut6P as substrate. These studies suggest that these isoforms could participated I different metabolic routesDoutoradoDoutor em Ciência

Purificação e caracterização das fosfatases acidas das sementes de soja quiescentes

Orientador: Hiroshi AoyamaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Quatro frações contendo atividade fosfatásica ácida (AP1, AP2, AP3A e AP3B) foram detectadas e purificadas à partir do extrato solúvel de sementes de soja quiescentes através da precipitação com sulfato de amônio e acetona e cromatografias de troca iônica, filtração em gel e afinidade. Em coluna de SP-Sephadex a fração APl foi eluída com o tampão de equilíbrio (tampão acetato 0,01 M, pH 5,0), enquanto as frações AP2 e AP3 foram eluídas com 0,05 e 0,3 M de fosfato, respectivamente. As frações AP3A e AP3B foram obtidas da fração AP3 aplicada em coluna de DEAE-Sephadex. A atividade enzimática foi determinada utilizando p-NPP como substrato em tampão acetato 100 mM, pH 5,0, a 37°C. Atividades específicas de 50, 10, 0,84 e 2 'um¿ .min-1.mg-1 foram obtidas para Apl (purificação de 910 vezes), AP2 (180 vezes), AP3A (16 vezes), e AP3B (36 vezes), respectivamente. Massas moleculares relativas de 51.000, 58.000, 52.000 e 30.000 foram determinadas para APl, AP2, AP3A e AP3B, respectivamente, através de filtração em gel na coluna SW 300 (Waters Protein Glass) acoplada a um sistema de LC. Das 4 frações, somente a APl se ligou à coluna de conA-Sepharose, embora provavelmente as outras frações também tenham carboidratos em suas estruturas. O perfil de eluição nas cromatografias de troca iônica mostrou que as 4 frações apresentavam pI diferentes. Após a purificação, foram realizados estudos cinéticos para as 4 frações de fosfatases ácidas. A fração APl apresentou uma alta atividade em pH 4,5-6,0, enquanto para as outras frações, a faixa de pH ótimo foi 5,0-6,5. APl e AP2 exibiram maior especificidade pelo substrato do que AP3A e AP3B. Os valores de Km foram determinados para p-NPP, Tyr-P e PPi, em pH 5,0 a 37°C. As fosfatases ácidas apresentaram os seguintes valores de Km: APl (p-NPP - 0,49, PPi - 0,51 e Tyr-P - 1,14 mM); AP2 (p-NPP - 0,38, PPi - 1,33 e Tyr-P - 1,14 mM); AP3A (p- NPP - 0,20, PPi - 0,16 e Tyr-P - 0,19 mM) e AP3B (p-NPP - 0,086, PPi - 0,17 e Tyr-P - 0,17 mM). A atividade das 4 frações não foi afetada na presença de EDTA, DTT, GSH e 2-mercaptoetanol. O fosfato, fluoreto, vanadato, molibdato e os cátions CU2+e Zn2+,inibiram as 4 frações, quando se utilizou o p-NPP como substrato. Ao contrário de outras fosfatases ácidas de planta, as 4 frações apresentaram alta atividade em temperatura acima de 80°C, durante 10 minutos de incubação. No entanto, quando as mesmas foram pré-incubadas a 80°C houve perda da atividade após 5 min., indicando portanto que a presença do substrato é importante para a manutenção da atividade nesta temperatura. Nenhuma das frações catalizou a reação de transfosforilação, uma vez que na presença de aceptores de fosfato (glicerol, metanol e etanol) não ocorreu aumento da atividade enzimática. Os resultados obtidos mostram que as 4 frações não apresentaram diferenças significativas em suas propriedades cinéticasAbstract: Four fractions of acid phosphatase (API, AP2, AP3A,and AP3B) have been detected and purified from soybean seeds soluble extract through ammonium sulfate and acetone precipitations, and ion exchange, gel filtration and concanavalin Asepharose chromatographies. API was eluted with equilibrium buffer, while AP2 and AP3 were eluted with 0.05 and 0.3 M inorganic phosphate, respectively, from SP-Sephadex column, AP3A and AP3B were the enzymatic fractions originated from AP3 applied on the DEAE-Sephadex column. The enzyme activity was determined using p-nitrophenylphosphate as substrate in 100mM acetate buffer, pH 5, at 37°C. Specific activity values of 50, 10, 0.84 and 2 Umol.min-1.mg-w1 ere obtained for API (910-fold purification), AP2 (180-fold), AP3A (16-fold), and AP3B (36-fold), respectively.The relative molecular mass of API, AP2, AP3A and AP3B were determined by 300 SW Waters Protein Glass column, were found to be 51,000, 58,000, 52,000 and 30,000, respectively. The four acid phosphatase forms purified seemed to be glycoproteins, however, only AP1 could bind to concanavalina A Sepharose. Ion exchange chromatography elution pattems showed that soybean seed acid phosphatases essentially differed in relation to their isoelectric point. The kinetic properties of the four fractions of soybean seeds acid phosphatases were studied. API presented a high activity at pH 4.5-6.0, while for the other fractions the optimum pH range was 5.0-6.5. API and AP2 exhibited higher substrate specificity than AP3A and AP3B. The Km values were determined for p-nitrophenylphosphate, tyrosinephosphate and inorganic pyrophosphate, at pH 5.0 and 37°C. The acid phosphatases presented the following apparent Km values: API (pNPP -0.49, PPi -0.51 and TyrP - 1.14mM);AP2 (pNPP- 0.38, PPi - 1.33 and TyrP - 1.14 mM); AP3A (pNPP - 0.20, PPi - 0.16 and TyrP - 0.19 mM) and AP3B (pNPP - 0.086, PPi - 0.17 and TyrP - 0.17 mM). Using pNPP as substrate, no effect was observed in the acid phosphatases activity in the presence of EDTA, dithiothreitol, glutathione and 2-mercaptoethanol. The four fractions were inhibited by inorganic phosphate, fluoride, vanadate, molybdate, CU2+and Zn2+.In contrast to other plant acid phosphatases alI the soybean seeds enzymatic forms presented high activities above 80°C, during 10 min incubation. However the enzymes were inactivated after 5 min when pre-incubated at 80°C in the absence of substrate. The soybean seeds acid phosphatases did not catalyze the transphosphorylation reaction since no stimulation was observed with inorganic phosphate acceptors (glycerol, methanol and ethanol). Our results showed that no significative differences could be observed on the kinetic properties for the four soybean seed acid phosphatase fractionsMestradoBioquimicaMestre em Ciências Biológica

Acid Phosphatase Activities During The Germination Of Glycine Max Seeds.

In this paper, we describe a study concerning the determination of some characteristics of soybean seedlings and the detection of acid phosphatase activities towards different substrates during the germination. Enzyme activities with p-nitrophenylphosphate (pNPP) and inorganic pyrophosphate (PPi) as substrates were detected from the 5th and 7th days after germination, respectively. Acid phosphatase activities with tyrosine phosphate (TyrP), glucose-6-phosphate (G6P) and phosphoenol pyruvate (PEP) were also observed but to a lesser extent. Under the same conditions, no enzyme activity was detected with phytic acid (PhyAc) as substrate. The appearance of phosphatase activity was coincident with the decrease of inorganic phosphate content during germination; over the same period, the protein content increased up to the 5th day, decreased until the 8th day, and remained constant after this period. Relative to phosphatase activity in the cotyledons, the activities detected in the hypocotyl and roots were 82% and 38%, respectively. During storage the enzyme maintained about 63% of its activity for 3 months at 5 degrees C. The specificity constant (Vmax/Km) values for pNPP and PPi were 212 and 64 mu kat mM-1 mg-1, respectively. Amongst the substrates tested, PPi could be a potential physiological substrate for acid phosphatase during the germination of soybean seeds.4215-2

Mecánica terrestre: humanismo y modernidad en la primera poesía de Carmen Conde (1929-1939)

La obra poética de Carmen Conde recoge las aportaciones literarias más relevantes de su época. Desde 1929 hasta 1939, la autora asume un simbolismo personal que se proyecta sobre la representación impresionista de la naturaleza (Brocal), la intensidad imaginativa asociada con los objetos cotidianos (Júbilos) o la experiencia trágica de la Guerra Civil (Mientras los hombres mueren). Su concepción lírica se defi ne por compatibilizar los signos externos de la modernidad con un humanismo que atraviesa todas las facetas de una vida dedicada a la escritur

Proteínas tirosina fosfatases: propriedades e funções biológicas Protein tyrosine phosphatases: properties and biological functions

<abstract language="eng">Protein phosphorylation-dephosphorylation catalyzed by the opposing and dynamic action of protein kinases and phosphatases probably, is the most crucial chemical reaction taking place in living organisms. Protein phosphatases are classified according to their substrate specificity and sensitivity to inhibitory or activator agents, into two families of protein phosphatases: serine/threonine phosphatases and tyrosine phosphatases (PTPs). PTPs can be divided into 3 groups: tyrosine specific phosphatases, dual and low molecular weight phosphatases. The role of tyrosine phosphorylation in mitogenic signaling is well documented, and one would predict that vanadate, pervanadate and other oxidant agents (protein tyrosine phosphatase inhibitors) may act as a growth stimulator

Biological behavior of pre-osteoblasts on natural hydroxyapatite: A study of signaling molecules from attachment to differentiation

8 p. : il., tab.Several biomaterials have been widely used in bone regeneration in both orthopedic and oral surgeries. However, it is poorly understood how these biomaterials alter osteoblast phenotype. It prompted us to examine the involvement of signaling proteins during preosteoblast adhesion (attachment), proliferation, and differentiation on natural hydroxyapatite (HA) from bovine bone. Our results indicated that natural HA is able to promote osteoblast adhesion, proliferation, and differentiation. The osteoblast/HA interaction requires phosphorylation of tyrosine residues of focal adhesion kinase, Src, and Paxillin upon integrin activation, which culminates in the control of cofilin phosphorylation (at serine 03) via rac-1 activation. In part, these signaling pathways are responsible for actin-rearrangement, responsible to adapt cell-shape on HA particles. In regarding to osteoblast differentiation, we showed that natural HA favored extracellular matrix remodeling by stimulating matrix metalloproteinase activities and alkaline phosphatase activity. Overall, this study demonstrates that osteoblast response toward bovine bone HA is initially mediated by activation of focal adhesion components, culminating on actin-rearrangement executed by cofilin activation via rac-1. Moreover, bovine bone HA provided an excellent microenvironment for osteoblast activity, since adhesion to differentiation